CN108588252A - A kind of parasite detection kit and its detection method - Google Patents
A kind of parasite detection kit and its detection method Download PDFInfo
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- CN108588252A CN108588252A CN201810272384.0A CN201810272384A CN108588252A CN 108588252 A CN108588252 A CN 108588252A CN 201810272384 A CN201810272384 A CN 201810272384A CN 108588252 A CN108588252 A CN 108588252A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to parasite detection field, a kind of parasite detection kit and its detection method are disclosed.A kind of parasite detection kit, include one or more nucleic acid amplification primers pair in the nucleic acid reaction system of kit, for expanding trypanosome, Leishmania, leishmania brasiliensis, chagasi Leishmanias, schizotrypanum cruzi, Lang Jieershi trypanosomes, Leishmania, leishmania mexicana or Leishmania amazonensis, panamensis Leishmania donovanis, guyanensis Leishmanias, or the nucleotide sequence of the leishmanial DNA of peruviana, detection method is using one or more nucleic acid amplification primers to carrying out nucleic acid amplification reaction, and the presence by detecting the DNA of the specific amplicon detection parasite of nucleic acid amplification primers pair.Compared with PCR, the amplification mode of RPA to diagnose is carried out in the simple equipment of constant temperature, low cost, high sensitivity, can be used for trypanosome, Leishmania mankind's early infection and the dog of infection identification, to reduce the VL incidence in human body.
Description
Technical field
The present invention relates to parasite detection field more particularly to a kind of parasite detection kits and its detection method.
Background technology
Leishmaniasis is the disease for influencing more than 88 countries notable global implication in the world, causes leishmaniasis
Protozoan is Leishmania.Cutaneous Leishmaniasis (CL) is characterized in chronic ulcer of skin, may influence the function of individual
State causes expensive and is not treated in time, and leads to scar of disfeaturing.Generally speaking, there are ten thousand leishmaniasis of 10-20 in the whole world.
Leishmaniasis causes to include local skin leishmaniasis disease (LCL), the oropharynx of destructive nasal cavity and leishmaniasis (ML)
The frequency spectrum of lesion.LCL is most common Viannia subgenus kind (L. (V.) braziliensis, L. (V.) panamensis, L.
(V.) guyanensis, L. (V.) peruviana), and cause by Leishmania subgenus kind lesser extent (L. (V.) Mo Xi
Brother, L. (V.) Amazon).ML is usually by L.braziliensis and less frequently by L. (V.) panamensis or L. (V.)
Caused by guyanensis.
It is that this disease occurs in the limited area of resource in the significant challenge of the diagnosis of leishmaniasis.Parasite at present
Learning diagnosis must be arranged in hospital, the condition that most of patients does not diagnose.In military field work and training, advanced experiment
Room equipment and medical worker are rare.For CL or ML, it is necessary that skin (skin) or mucosal tissue or lesion biopsy punching, which scrape,
, the sensibility through Tissue pathological diagnosis, it is low (40-70%) that success rate is observed in smear culture under the microscope.Based on antibody
Any clinical forms of test leishmaniasis be useful, realized using PCR, but of high cost, in the area of natural resources shortage
Implement not real enough.
Therefore other method detection infectious diseases are needed, method needs quickly, inexpensively, specific and sensitive, does not need to be high
Expensive equipment or the equipment for being susceptible to failure.
Present inventors have developed can detect the recombinase polymeric enzymatic amplification of trypanosome in the biological sample based on it
(RPA) the sensitive isothermal duplication experiment of one under method.Trypanosome is by only having single flagellum to distinguish one group of kinetoplast protozoan.
Trypanosome is related to the life cycle of vertebrate, invertebrate or plant.It is former that Oligonucleolide primers are designed to amplification Li Shiman
Worm or trypanosome nucleic acid, primer are directed into the DNA (kDNA) of kinetoplast.Kinetoplast as used herein is comprising mitochondria
The network of the big Intramitochondrial cyclic DNA (being known as kDNA) of many copies of genome.Kinetoplasts only moves base in class
Body mesh protozoan finds.One kinetoplast is typically the flagellar basal body of adjacent organism, this shows that it combines closely
Cytoskeleton.Primer is designed to make their not other leishmanias or the horizontal amplification of nucleic acid (for example, kDNA) of trypanosome.
By using the subject that one of these primers can identify, such as people or dog, specific parasite has been infected (for example, baby is sharp
Assorted graceful protozoon) there is the possibility of the related protozoon parasite of overlapping host range to the detection excluded.
Recombinase polymeric enzymatic amplification (RPA) is that a single tube isothermal substitutes polymerase chain reaction (PCR).It is reversed by being added
The RPA reactions of record enzyme can detect RNA and DNA, without an independent step, to generate cDNA's.Because it is
Isothermal, RPA reactions need the equipment more much simpler than PCR.RPA reactions in theory can be quickly and easily by keeping managing
Operation.This so that RPA is developing low-cost, quickly, the ideal chose of point-of-care molecular testing.It is needed with conventional PCR high
Expensive equipment is compared, and RPA is carried out in steady temperature, uncomplicated equipment, may be used as low cost, and the diagnosis of high sensitivity
Test, with reduce or improve the control program of the VL of the electric current of public health agencies validity interruption Leishmania (e.g.,
Leishmania infantum), with the transmission of human colony, this is the identification for being conceived to mankind's early detection and infected dog.Dog
The main source of the mankind of infection by Leishmania infantum, infected animal, and then removed from sending cycle it is accurate
Identification is one of most effective strategy, to reduce the VL incidence in human body.
Kit reaction described here mixture includes one or more recombinases, single-stranded DNA binding protein
(SSB), strand displacement polymerase.Kit reaction mixture further may further include exonuclease
In (for example, circumscribed III), endonuclease (IV) or reverse transcriptase.In addition to the kit of enzyme reaction mixing may include that fluid infusion is slow
Electuary, reaction buffer, primer, label, detection reagent etc..It can pass through addition as the RPA reactions using PCR, form of ownership
Further primer/probe allows the detection of multiple analytes or in same pipe internal contrast to being re-used.
It is less complex compared to ring mediated isothermal amplification (LAMP) or other isothermal amplification methods by RPA isothermal duplications, and
And lateral flow detection can be easily adaptable.Nearest publication has confirmed the applicability of this technology, to detect disease
Poison and bacterium infection.Approach described herein uses parasite under conditions of RPA detection isothermal duplications.
Invention content
The present invention is directed to the shortcomings that needing expensive equipment in the prior art, provide a kind of parasite detection kit and its
Detection method.
In order to solve the above-mentioned technical problem, the present invention is addressed by following technical proposals：
Parasite detection kit, the interior nucleic acid reaction system of kit includes a kind of nucleic acid amplification primers pair, including with
It is at least one of lower：
(A) nucleotide sequence of the DNA of trypanosome can be expanded including at least one kind in following nucleotide sequences：(1)SEQ
ID NO：26, SEQ ID NO：27, SEQ ID NO：One or more and SEQ ID NO in 28：23；(2)SEQ ID NO：
26, SEQ ID NO：27, SEQ ID NO：One or more and SEQ ID NO in 28：24；(3)SEQ ID NO：26, SEQ
ID NO：27 or SEQ ID NO：One or more and SEQ ID NO in 28：25；
(B) nucleotide sequence of leishmanial DNA can be expanded including at least one kind in following nucleotide sequences：
(1)SEQ ID NO：6, SEQ ID NO：7, SEQ ID NO：One or more and SEQ ID NO in 8：3；(2)SEQ ID
NO：6, SEQ ID NO：7, SEQ ID NO：One or more and SEQ ID NO in 8：4；(3)SEQ ID NO：6, SEQ ID
NO：7, SEQ ID NO：One or more and SEQ ID NO in 8：5；(4)SEQ ID NO：12, SEQ ID NO：In 13
One or more and SEQ ID NO：10；(5)SEQ ID NO：12, SEQ ID NO：One or more and SEQ ID in 13
NO：11；
(C) nucleotide sequence that can expand the DNA of leishmania brasiliensis includes at least in following nucleotide sequences
It is a kind of：(1)SEQ ID NO：19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：16；
(2)SEQ ID NO：19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：17；(3)SEQ
ID NO：19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：18；
(D) nucleotide sequence that can expand the leishmanial DNA of chagasi includes at least in following nucleotide sequences
One kind：(1)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：One or more in 42 and
SEQ ID NO：32；(2)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：One kind in 42 or
A variety of and SEQ ID NO：33；(3)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：In 42
One or more and SEQ ID NO：34；(4)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：
One or more and SEQ ID NO in 42：35；(5)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ
ID NO：One or more and SEQ ID NO in 42：36；(6)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：
41, SEQ ID NO：One or more and SEQ ID NO in 42：38；
(E) nucleotide sequence of the DNA of schizotrypanum cruzi can be expanded：SEQ ID NO：50 and SEQ ID NO：51；
(F) nucleotide sequence of the DNA of schizotrypanum cruzi can be expanded：SEQ ID NO：53 and SEQ ID NO：54；
(G) nucleotide sequence of the DNA of Lang Jieershi trypanosomes can be expanded including at least one in following nucleotide sequences
Kind：(1)SEQ ID NO：56, SEQ ID NO：57, SEQ ID NO：One or more and SEQ ID NO in 58：59；(2)
SEQ ID NO：56, SEQ ID NO：57, SEQ ID NO：One or more and SEQ ID NO in 58：60；
(H) the nucleotide sequence composition of the DNA of Lang Jieershi trypanosomes can be expanded：SEQ ID NO：63 and SEQ ID NO：
64；
(I) the nucleotide sequence composition of leishmanial DNA can be expanded：SEQ ID NO：66, SEQ ID NO：67
In one or more and SEQ ID NO：68；
(J) the nucleotide sequence composition of the DNA of leishmania mexicana or Leishmania amazonensis can be expanded：
SEQ ID NO：70 and SEQ ID NO：71；
(K) leishmania brasiliensis, panamensis Leishmania donovanis, guyanensis Li Shiman originals can be expanded
The nucleotide sequence of worm or the leishmanial DNA of peruviana form：SEQ ID NO：73, SEQ ID NO：One in 74
Kind is a variety of；
Preferably, in step A-K, the nucleic acid probe group of Leishmania or trypanosome DNA fragmentation for detecting amplification
At including：SEQ ID NO：9, SEQ ID NO：14, SEQ ID NO：77, SEQ ID NO：29, SEQ ID NO：30, SEQ ID
NO：37, SEQ ID NO：52, SEQ ID NO：55, SEQ ID NO：61, SEQ ID NO：62, SEQ ID NO：65, SEQ ID
NO：69, SEQ ID NO：72：
Preferably, with nucleic acid probe detect labelling kit, nucleic acid probe be include nucleic acid sequence SEQ ID NO：76
Oligonucleotides.
Preferably, further include amplifing reagent, reagent, one or more recombinases, endonuclease, an excision enzyme,
Single-stranded DNA binding protein or strand displacement polymerase；
Preferably, exonuclease is exonuclease III, endonuclease is a kind of endonuclease IV.
Preferably, including the nucleic acid sequence for the oligonucleotides nucleic acid probe that can detect at least one label：SEQ ID
NO：9, SEQ ID NO：14, SEQ ID NO：77, SEQ ID NO：29, SEQ ID NO：30, SEQ ID NO：37, SEQ ID
NO：52, SEQ ID NO：55, SEQ ID NO：61, SEQ ID NO：62, SEQ ID NO：65, SEQ ID NO：69, SEQ ID
NO：72, SEQ ID NO：76.
Preferably, including one or more nucleic acid amplification primers in the parasite detection kit using claim 1
To carrying out nucleic acid amplification reaction, and depositing by the DNA of the specific amplicon detection parasite of detection nucleic acid amplification primers pair
.
The method for detecting the specific amplicon of nucleic acid amplification primers pair includes being visited with the nucleic acid comprising following nucleic acid sequence
The amplification of nucleic acid that needle combines：SEQ ID NO：9, SEQ ID NO：14, SEQ ID NO：77, SEQ ID NO：29, SEQ ID
NO：30, SEQ ID NO：37, SEQ ID NO：52, SEQ ID NO：55, SEQ ID NO：61, SEQ ID NO：62, SEQ ID
NO：65, SEQ ID NO：69, SEQ ID NO：72, SEQ ID NO：76.
Preferably, further including forming the mixing comprising at least one primer pair and the biological sample for participating in amplified reaction
Object, biological sample are body fluid or tissue sample.Sample source includes tissue, such as biopsy and autopsy samples, and takes histology purpose
Freezing microtome section slice.Body fluid includes blood and blood constitutent or product (for example, serum, blood plasma, blood platelet, red blood cell
Deng), saliva, phlegm, excrement, urine etc..Biological sample is typically to be obtained from eucaryote, most preferably mammal such as primate
Such as chimpanzee, people, ox, dog, cat, rodent, for example, cavy, rat, mouse, rabbit, bird, reptile, insect.
Preferably, amplified reaction is the isothermal amplification of a recombinase polymeric enzymatic amplification (RPA).
A " target sequence " or " target nucleic acid sequence " refer to as described in this article as used herein or it is complementary, is put
Greatly, it detects, or one or more polynucleotides that the nucleic acid sequence of amplification and the parasite detected uses simultaneously.In addition, though
Term target sequence is sometimes referred to as a double-strandednucleic acid sequence, but target sequence can also be single-stranded.It is the feelings of double-strand in target
Under condition, polynucleotide primers sequence of the invention will preferably expand two chains of target sequence.Target sequence can be by specific organism
It is more or less specific.For example, target sequence can be specific for entirely belonging to, multiple categories, a species or subspecies, sero-group, serum
Other subsets of type, strain, separation or organism.The present invention polynucleotide sequence be selected as they specially with a series of Asias
Kind or serotype, the ability of the hybridization of trypanosome.
As used herein, " separation " word can refer to a nucleic acid or oligonucleotides ancestral home source (when passing through recombinant DNA
Technology produce), it be substantially free of cellular material, bacterial material, viral material or culture medium or precursor or other
Chemicals (when chemical synthesis).Moreover, " nucleic acid fragment of separation " or " oligonucleotides of separation " is nucleic acid or oligonucleotides,
It is not naturally occurring.
In some aspects, it is expanded before nucleic acid sequence is detected." amplification " word is defined from nucleic acid molecule
Single or rudimentary copy number makes the process of multiple copies of nucleic acid.The nucleic acid of amplification can be referred to as amplicon.
Such as oligonucleotides or amplicon can be labeled, that is, are conjugated or are covalently or non-covalently connected to other parts, such as
Protein, peptide, carrier, fluorescence part or label.Term " conjugate " or " immune " be widely used to define part with
Another effective combination of drug, it is no intended to it is associated only to refer to any kind of operation, it is not particularly limited, with
Chemistry is " conjugated." nucleic acid or oligonucleotides of separation in some aspects are conjugated with detectable label.
Term " label " about probe be intended to by with another reagent be direct-coupling (that is, physical connection) probe can
Detection substance to probe and indirect labelling surrounds the direct label label of probe by reacting.The example of indirect labelling includes
Use the antibody and end mark or biotin of fluorescent marker so that it can use the strepto- of fluorescent marker to resist detected DNA
The detection of the probe of the central marks of probe.
" label ", " detectable label " or " detectable part " are by spectroscopy, photochemistry, biochemistry, immunization
It learns, the composition of chemistry or the detection of other physical means.For example, useful label includes fluorescent dye, and electron-dense reagents, enzyme
(for example, common in ELISA), biotin, foxalin or haptens and protein, can be made into detectable, example
Such as, it is reacted with peptide specific by mixing antibody of the radioactive label at peptide or for detection.
Dyestuff is covalently attached to the ends 5' of oligonucleotide probe such as FAM dyestuffs.Other dyestuff examples include such as
TAMRA, AlexaFluor dyestuff such as AlexaFluor 495 or 590, waterfall is blue, blue strand, and the Pacific Ocean is blue, and Oregon is green,
Rhodamine, fluorescein, TET, HEX.It each can be by being quenched in the ends 3' or other non-fluorescence quencher dyes.Quenching point
Son is suitably matched to the dyestuff of maximum fluorescence.Any suitable fluorescence probe for being used in nucleic acid amplification detecting system is
In exemplary operation of the present invention.
" specific binding " refers to presence of the conclusive molecule in the heterogeneous population of other biological agents to target
Existing association reaction.Therefore, under specified determination condition, specified molecule preferentially combines particular target, and in significant quantity
Other biological product present in sample does not combine.
The present invention has significant technique effect as a result of above technical scheme：Expand Zeng Fangfa with traditional PCR to set
Standby expensive, of high cost to compare, the amplification mode of RPA to diagnose is carried out in constant temperature simple equipment, inexpensive, sensitivity
Height, can be used for trypanosome, Leishmania mankind's early infection and the dog of infection identification, with reduce human body VL fall ill
Rate.
Description of the drawings
Fig. 1 is different kinetoplast parasite specific amplification figures.(several groups of primers are designed to expand the small ring of kinetoplast
DNA specifically expands leishmania infantum, the inside region of schizotrypanum cruzi, leishmania brasiliensis.Specific parasite DNA
(specificity of amplification is by the way that with different DNA profilings, (the 1st swimming lane is confirmed including the primer is cultivated for target：Baby Li Shi
Graceful protozoon；2nd swimming lane：Schizotrypanum cruzi, the 3rd swimming lane：Leishmania brasiliensis), incoherent target DNA (the 4th swimming lane：The mankind；The
5 swimming lanes：Dog；6th swimming lane：Giardia lamblia) and primer (the 7th swimming lane) without template.In addition, the 8th swimming lane is the marker of 100bp)
Fig. 2 is risen violently sensitivity test RPA electrophoretograms from the various parasites of blood purification DNA.Prepare dilution DNA：Dog
Hepatic blood DNA is diluted to 100 microlitres, corresponds to, A dilutions 1：130000；B dilutions 1：32,500；C dilutions 1：8,
125；D dilutions 1：2031；E dilutions 1：508；F dilutions 1：127；G dilutions 1：32；H dilutions 1：8；Parasite) it uses
Make the template to RPA.Dilution A-D shows stronger amplification (not shown), and the amplification positive drops to parasite (the 1st swimming
Road).The primer of the label (MK) of no template (J) and 100bp is also shown.
Fig. 3 is and the basic RPA electrophoretograms of rangeli trypanosomes (panel).(draw：T.rangeli ITS1.Sample:Water,
T.rangeli, schizotrypanum cruzi, L.braziliensis, L.chagasi, people, dog and giardia lamblia.)
Fig. 4 is the basic RPA electrophoretograms of leishmania major (panel).(draw：Leishmania major.Sample:Water, it is large
Big Leishmania, L.braziliensis, L. Amazons, schizotrypanum cruzi, people, dog and giardia lamblia.)
Fig. 5 is and the basic RPA electrophoretograms of schizotrypanum cruzi (panel).(draw：Schizotrypanum cruzi.Sample：Water, Meraloo,
Tejapileo, T.rangeli, L.braziliensis, L. Amazon, L.chagasi, people, dog and giardia lamblia.)
Specific implementation mode
Present invention is further described in detail with embodiment below in conjunction with the accompanying drawings.
Embodiment 1
A kind of parasite detection kit expands specific post to amplimer or RPA primer specificities as shown in Figs. 1-5
Infested generation parasite specific amplicon.Probe and amplicon are specific amplification Leishmania donovani, Li Shiman
Chagasi, leishmania infantum.These parasite specific amplicons are detected with oligonucleotide probe.Probe is specially to set
It is calculated as between targeting unique DNA sequence dna, and passes through two primer amplifications.The specificity that the combination of primer and probe determines (should
DNA sequence dna will be detected) to test.In some aspects, Leishmania donovani, Li Shiman chagasi, leishmania infantum
Specificity amplification primer, including there is 90%, 95%, 98% or 100% identical oligonucleotide sequence, oligonucleotide sequence packet
Include SEQ ID NO：3,4,5,6,7,8,10,11,12 or 13.Amplimer includes：With 25,26,27,28,29,30,31,
32,33,34,35,36,37,38,39,40,41,42,43,44,45 continuous oligonucleotides.
Some aspects are related to amplimer to including SEQ ID NO：3/6,3/7,3/8,4/6,4/7,4/8,5/6,5/7,
5/8,10/12,10/13,11/12,11/13.RPA probes include oligonucleotides nucleotide sequence SEQ ID NO：9 or SEQ ID
NO：14.Further, SEQ ID NO：14 include 28 bit intervals that tetrahydrofuran (THF) replaces guanosine.
Embodiment 2
Primer, probe and amplicon are specific amplification leishmania brasiliensis.Oligonucleotide probe is to detect these to post
Infested specific amplicon.Leishmania brasiliensis amplimer includes having 70,75,80,85,90,95,98 or 100% phases
Same oligonucleotide sequence, oligonucleotide sequence includes SEQ ID NO：16,17,18,19,20,21.In some aspects, it expands
Primer includes：With 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45
Continuous oligonucleotides.Some aspects are related to amplimer to including SEQ ID NO：16/19,16/20,16/21,17/19,
17/20,17/21,18/19,18/20 or 18/21.The oligonucleotides SEQ ID NO for the nucleotide sequence that RPA probes include：
77。
Embodiment 3
Primer, probe and amplicon are specific amplification trypanosome trypanosome or trypanosome rangeli.Oligonucleotide probe is detection
These parasite specific amplicons.In some aspects, schizotrypanum cruzi amplimer includes 90,95,98 or 100% identical widows
Nucleotide sequence, oligonucleotide sequence include SEQ ID NO：23,24,25,26,27,28.Amplimer includes：With 25,
26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or 45 continuous oligonucleotides.
Some aspects are related to amplimer to including SEQ ID NO：23/26,23/27,23/28,24/26,24/27,24/28,25/
26,25/27 or 25/28.RPA probes may include the oligonucleotides of the nucleotide sequence with SEQ ID NO：T.rangeli
Trypanosome or SEQ ID NO：29,30.
Embodiment 4
Primer, probe and amplicon are specific amplification leishmania chagasi.Further, oligonucleotides is visited
Needle is designed to detect these parasite specific amplicons.The Leishmania chagasi amplimers include 90,95,98
Or 100% identical oligonucleotide sequence, oligonucleotide sequence includes SEQ ID NO：32,33,34,35,36,37,38,39,
40,41.Amplimer includes：With 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,
43,44 or 45 continuous oligonucleotides.Some aspects are related to amplimer to including SEQ ID NO：32/39,32/40,41
/ 32,33/32/42nds, 39,33/33/40ths, 41,34/33/42nds, 34/39,34/40,41,34/42,35/39,
35/35/40ths, 41,35/42,36/39,36/40,36/41,36/42,38/39,38/40,38/41 or 38/42.RPA probes
Including oligonucleotides SEQ ID NO：37.
Embodiment 5
Primer, probe and amplicon are specific amplification schizotrypanum cruzis.Oligonucleotide probe, which is designed to detect these, to be posted
Infested specific amplicon.Primer and probe can be designed to expand the subtelomeric area of trypanosome.Schizotrypanum cruzi amplimer packet
90,95,98 or 100% identical oligonucleotide sequences are included, oligonucleotide sequence includes SEQ ID NO：50,51.With 25,
26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or 45 continuous oligonucleotides.
Some aspects are related to amplimer to including SEQ ID NO：50/51.RPA probes may include the nucleosides for having SEQ ID NO
The oligonucleotides of acid sequence：52.
Embodiment 6
Primer, probe and amplicon are specific amplification schizotrypanum cruzis.Further, oligonucleotide probe is designed to examine
Survey these parasite specific amplicons.Primer and probe can be designed to expand the regions ITS1 of trypanosome.Schizotrypanum cruzi expands
It includes 90,95,98 or 100% identical oligonucleotide sequences to increase primer, and oligonucleotide sequence includes SEQ ID NO：5354.Expand
Increasing primer includes：With 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or
45 continuous oligonucleotides.Some aspects are related to amplimer to including SEQ ID NO：53/54.RPA probes may include
The oligonucleotides of nucleotide sequence with SEQ ID NO：55.
Embodiment 7
Primer, probe and amplicon are specific amplification trypanosome rangeli.Further, oligonucleotide probe
It is designed to detect these parasite specific amplicons.In some aspects, primer and probe can be designed to expand
The subtelomeric area of T.rangeli.In some aspects, trypanosome rangeli amplimers include 90,95,98 or 100% identical
Oligonucleotide sequence, oligonucleotide sequence include SEQ ID NO：56,57,58,5960.Amplimer includes：With 25,26,
27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or 45 continuous oligonucleotides.Certain
A little aspects are related to amplimer to including SEQ ID NO：56/59,57/59,58/59,56/60,57/60,58/60.RPA probes
It may include the oligonucleotides of the nucleotide sequence with SEQ ID NO：61 or 62.
Embodiment 8
Primer, probe and amplicon are specific amplification trypanosome rangeli.Further, oligonucleotide probe is designed
To detect these parasite specific amplicons.Primer and probe can be designed to expand the regions ITS1 of trypanosome rangeli.
Trypanosome rangeli amplimers include 90,95,98 or 100% identical oligonucleotide sequences, and oligonucleotide sequence includes
SEQ ID NO：63 or 64.Amplimer includes：With 25,26,27,28,29,30,31,32,33,34,35,36,37,38,
39,40,41,42,43,44 or 45 continuous oligonucleotides.Some aspects are related to amplimer to including SEQ ID NO：63/
64.RPA probes may include the oligonucleotides of the nucleotide sequence with SEQ ID NO：65.
Embodiment 9
Primer, probe and amplicon are specific amplification leishmania majors.Further, oligonucleotide probe is set
It is calculated as detecting these parasite specific amplicons.Primer and probe can be designed to expand area between leishmanial ITS1
Domain.Leishmania major amplimer includes 90,95,98 or 100% identical oligonucleotide sequences, oligonucleotide sequence packet
Include SEQ ID NO：66,67 or 68.In some aspects, amplimer includes：With 25,26,27,28,29,30,31,32,
33,34,35,36,37,38,39,40,41,42,43,44 or 45 continuous oligonucleotides.Some aspects are related to amplimer
To including SEQ ID NO：66/68 or 67/68.RPA probes may include the few nucleosides of the nucleotide sequence with SEQ ID NO
Acid：69.
Embodiment 10
Primer, probe and amplicon are specific amplification leishmania mexicana or Leishmania Amazon.Into one
Step, oligonucleotide probe is designed to detect these parasite specific amplicons.Primer and probe can be designed to expand
Increase the regions ITS1 of leishmania mexicana or the Amazons Li Shiman.Leishmania mexicana or the amplification of the Amazons Li Shiman
Primer includes 90,95,98 or 100% identical oligonucleotide sequences, and oligonucleotide sequence includes SEQ ID NO：70 or 71.Expand
Increasing primer includes：With 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or
45 continuous oligonucleotides.It is related to amplimer to including SEQ ID NO：70/71.RPA probes may include with SEQ
The oligonucleotides of the nucleotide sequence of ID NO：72.
Embodiment 11
Primer, probe and amplicon are specific amplification leishmania brasiliensis, Du Shi Li Shiman panamensis, Li Shiman
Guyanensis, Leishmania peruviana's.Further, oligonucleotide probe is designed to detect these parasites
Specific amplicon.Primer and probe can be designed to expand leishmania brasiliensis, Du Shi Li Shiman panamensis, profit
The regions ITS1 of assorted graceful guyanensis, Leishmania peruviana.Leishmania brasiliensis, Du Shi Li Shiman
Panamensis, Li Shiman guyanensis, Leishmania peruviana amplimers include 90,95,8 or 100% identical
Oligonucleotide sequence, oligonucleotide sequence includes SEQ ID NO：73,74 or 75.Amplimer includes：With 25,26,27,
28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or 45 continuous oligonucleotides.Certain sides
Face is related to amplimer to including SEQ ID NOS：73/75ths or 74/75ths.RPA probes may include having
The oligonucleotides of the nucleotide sequence of SEQ ID NO：76.
Embodiment 12
For detecting Leishmania or trypanosomal parasites in sample comprising using one or more nucleic acid primers to such as
The nucleic acid amplification reaction of progress described herein simultaneously detects existing for the DNA of parasite specific amplicon by detection primer
Method.Detection primer is to the nucleic acid sequence SEQ ID NO that specific amplicon includes the amplification of nucleic acid combined with nucleic acid probe：9,
14,77,29,30,37,52,55,61,62,65,69,72 or 76, wherein the probe shows institute to the combination of the DNA of amplification
State the presence of the parasite in sample.This method may further include：It is formed comprising at least one primer pair and is carried out before
Amplified reaction sample part reaction mixture.
Certain embodiments are related to identical with sequence 90,95,98 or 100%, and oligonucleotide sequence includes SEQ ID
NO：2,3,4,5,6,7,8,16,17,18,19,20,21,23,24,25,26,27,28,32,33,34,35,36,37,38,39,
40,41,42,43,44,45,46,47,48 or 49-77.
Embodiment 13
Li Shiman chagasi ((GI 394857980)；SEQ ID NO：1).
Primer includes：LchaFW1, GATCGAGGCTGACACAATTTGATGGCCTGTG (SEQ ID NO：3), 31nt,
GC%51.6, TM 64, HG, 3, SF 20；
Lcha FW1.1-TGGCCTGTGTAAATATATCTTACCTTAAGGTGGC(SEQ ID NO：4), 34nt, GC%
41.2, TM 60.7, HG1, SF20；
LchaFW3-TCTGTTTGGTTATGTTATGATCGAGGCTGAC(SEQ ID NO：5) area, 31nt, NCR, GC
41.9%, 59.8TM, HG6, SF 16；
Lcha RV2-CTACCCGGAGGACCAGAAAAGTTTGGGA(SEQ ID NO：6), 28 nucleotide, region CR,
GC%53.6, TM 63.2；
Lcha RV4-TTCGGGATTTTCCACCAATTCCCAACCATC(SEQ ID NO：7), 30 nucleotide, GC contain
Measure 46.7, TM 62.5；
Lcha RV4.5-ACCAATTCCCAACCATCCAACACGTCCCAA(SEQ ID NO：8), 30 nucleotide,
GC%50, TM 65.1, HG4, SF 9.(HG=hairpin, SF=selfdimer)
Embodiment 14
Leishmania brasiliensis kinetoplast small ring (GI | 643458 | GB | U19803.1；SEQ ID NO：15).
Primer includes：LBRA 10-FW-TGCGCAGAGTTATTATGTTGTCAATGAATAA(SEQ ID NO：16),
31nt, GC%32.3, TM 57.2, HG10, SF 25；
LBRAFW20-GATGAAAATGTACTCCCCGACATGCCTCTG(SEQ ID NO：17), 30nt, GC%50, TM
62, HG2, SF 17；
LBRAFW25-AATTTGATGAAAATGTACTCCCCGACATGC(SEQ ID NO：18), 30nt, GC%40, TM
59.4, HG3, SF 20；
LBRARV30-TGAGACGGGGTTTCTGTATGCCATTTTTCGGT(SEQ ID NO：19), 31nt, GC%
45.2, TM 63.4, HG1, SF 18；
LBRARV40-CTAATTGTGCACGGGGAGGCCAAAAATAGCGA(SEQ ID NO：20), 32nt, GC%50,
TM 64.9, HG2, SF 20；
LBRARV45-AACCCCTAATTGTGCACGGGGAGGCCAAA(SEQ ID NO：21), 29nt, GC%55.2, TM
67.1, HG1, SF 19.
Embodiment 15
The small ring sequence of Y plants of dynamic bases of schizotrypanum cruzi clones M835, ORF, (SEQ ID NO：22).
Primer includes：Trypanosome -60-FW-AGCCTTATCACCCTTACCACCCACAGCCTA (SEQ ID NO：23),
30nt, GC%53.3, TM 65.2, HG2, SF14；
Tcruzi-65-FW-CAGCCTATATTACACCAACCCCAATCGAAC(SEQ ID NO：24), 30nt, GC%
46.7, TM 60.5, HG2, SF 15；
T.cruzi-70-FW-ACCCTTACCACCCACAGCCTATATTACACCA(SEQ ID NO：25), 31nt, GC%
48.4, TM 63.3, HG3, SF14；
Tcruzi-80-RV-GCGTTCAACTTTTGGGGCCCAGAATCATGC(SEQ ID NO：26), 30nt, GC%
53.3,65.1, HG4, SF19；
Trypanosome -85-RV-AACTTTTGGGGCCCAGAATCATGCATCTCC (SEQ ID NO：27), 30nt, GC%50,
TM 64, HG4, SF20；
Trypanosome -90-RV-CTGCAAGAAACTGGAAAATATGGTTTTGGGAG (SEQ ID NO：28), 32nt, GC%
40.6, TM 59.9, HG2, SF19.
Embodiment 16
Leishmania chagasi kinetoplast minicircle dnas, complete sequence (SEQ ID NO：31).
Primer includes：
LDOCHIN-FW100-TAGCCATAGCGCTTTAGAATAGTTCGRCTCCGAA(SEQ ID NO：32), 34nt,
GC 45.6Tm are 63.5；
LDOCHIN-FW110-TATAGCCATAGCGCTTTAGAATAGTTCGRCTCCGA(SEQ ID NO：33), 35nt,
GC 44.3, TM 62.9；
LDOCHIN-FW120-TATAGCCATAGCGCTTTAGAATAGTTCG(SEQ ID NO：34), 28nt, GC
39.3, TM 56.3；
LDOCHIN-FW130-TRCTCGTACACTATAAGTATTATGTTTAATATAT(SEQ ID NO：35), 34nt,
GC 22.1, TM 52.1；
LDOCHIN-FW140-CTGACATTRCTCGTACACTATAGTATTATG(SEQ ID NO：36), 31nt, GC
33.9, TM 54.2；
TAATAACTFACATTACTCGTACACTATAAGTATTATGTTTAATAT(SEQ ID NO：37)；
LDOCHIN-FW150-TAGTAATATCTRTACCGATATATTTRTAGGTTG(SEQ ID NO：38)；
LDOCHIN-RV160-CAACCTAYAAATATATCGGTAYAGATATTACTA(SEQ ID NO：39), 33nt, GC
27.3, TM 52.6；
LDOCHIN-FW170-CATACTGCAGTGAATTGRAAATTAATRAATTGG(SEQ ID NO：40), 33nt, GC
30.3TM 55.9；
LDOCHIN-RV180-CCAATTYATTAATTTYCAATTCACTGCAGTATG(SEQ ID NO：41)；
LDOCHIN-RV190-CCCTACCCGGAGGACCAGAAAAGTTTGG(SEQ ID NO：42), 28nt, GC 57.1, TM
63.9；
Probe CCAAACTTTTCTGGTCCTCCGGGTAGGGGCGTTCTGCAAA (SEQ ID NO：43).
Embodiment 17
Other primers include GAAAAATGGGTCCAGAAATGCCGTTCAA (SEQ ID NO：44)；
TTGAACGGGATTTCTGCACCCATTTTTC(SEQ ID NO：45)；
AAACTGGGGGTTGGTGTAAAATAGGGCCGG(SEQ ID NO：46)；
CCGGCCCTATTTTACACCAACCCCCAGTTT(SEQ ID NO：47)；
GGAAACTGGGGGTTGGTGTAAAATAGGGC(SEQ ID NO：48)；
GCCCTATTTTACACCAACCCCCAGTTTCC(SEQ ID NO：49)
In short, the foregoing is merely presently preferred embodiments of the present invention, it is all according to impartial made by scope of the present invention patent
Variation and modification, should all belong to the covering scope of patent of the present invention.
Claims (10)
1. a kind of parasite detection kit, which is characterized in that include one or more cores in the nucleic acid reaction system of kit
Sour amplimer pair, including it is at least one of following：
(A) nucleotide sequence of the DNA of trypanosome can be expanded including at least one kind in following nucleotide sequences：(1)SEQ ID
NO：26, SEQ ID NO：27, SEQ ID NO：One or more and SEQ ID NO in 28：23；(2)SEQ ID NO：26,
SEQ ID NO：27, SEQ ID NO：One or more and SEQ ID NO in 28：24；(3)SEQ ID NO：26, SEQ ID
NO：27 or SEQ ID NO：One or more and SEQ ID NO in 28：25；
(B) nucleotide sequence of leishmanial DNA can be expanded including at least one kind in following nucleotide sequences：(1)SEQ
ID NO：6, SEQ ID NO：7, SEQ ID NO：One or more and SEQ ID NO in 8：3；(2)SEQ ID NO：6, SEQ
ID NO：7, SEQ ID NO：One or more and SEQ ID NO in 8：4；(3)SEQ ID NO：6, SEQ ID NO：7, SEQ
ID NO：One or more and SEQ ID NO in 8：5；(4)SEQ ID NO：12, SEQ ID NO：It is one or more in 13
With SEQ ID NO：10；(5)SEQ ID NO：12, SEQ ID NO：One or more and SEQ ID NO in 13：11；
(C) nucleotide sequence of the DNA of leishmania brasiliensis can be expanded including at least one kind in following nucleotide sequences：
(1)SEQ ID NO：19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：16；(2)SEQ
ID NO：19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：17；(3)SEQ ID NO：
19, SEQ ID NO：20, SEQ ID NO：One or more and SEQ ID NO in 21：18；
(D) nucleotide sequence of the leishmanial DNA of chagasi can be expanded including at least one in following nucleotide sequences
Kind：(1)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：One or more and SEQ in 42
ID NO：32；(2)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：It is one or more in 42
With SEQ ID NO：33；(3) SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：One in 42
Kind or a variety of and SEQ ID NO：34；(4)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID NO：42
In one or more and SEQ ID NO：35；(5)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41, SEQ ID
NO：One or more and SEQ ID NO in 42：36；(6)SEQ ID NO：39, SEQ ID NO：40, SEQ ID NO：41,
SEQ ID NO：One or more and SEQ ID NO in 42：38；
(E) nucleotide sequence of the DNA of schizotrypanum cruzi can be expanded：SEQ ID NO：50 and SEQ ID NO：51；
(F) nucleotide sequence of the DNA of schizotrypanum cruzi can be expanded：SEQ ID NO：53 and SEQ ID NO：54；
(G) nucleotide sequence of the DNA of Lang Jieershi trypanosomes can be expanded including at least one kind in following nucleotide sequences：
(1)SEQ ID NO：56, SEQ ID NO：57, SEQ ID NO：One or more and SEQ ID NO in 58：59；(2)SEQ
ID NO：56, SEQ ID NO：57, SEQ ID NO：One or more and SEQ ID NO in 58：60；
(H) the nucleotide sequence composition of the DNA of Lang Jieershi trypanosomes can be expanded：SEQ ID NO：63 and SEQ ID NO：64；
(I) the nucleotide sequence composition of leishmanial DNA can be expanded：SEQ ID NO：66, SEQ ID NO：In 67
One or more and SEQ ID NO：68；
(J) the nucleotide sequence composition of the DNA of leishmania mexicana or Leishmania amazonensis can be expanded：SEQ ID
NO：70 and SEQ ID NO：71；
(K) leishmania brasiliensis can be expanded, panamensis Leishmania donovanis, guyanensis Leishmanias,
Or the nucleotide sequence composition of the leishmanial DNA of peruviana：SEQ ID NO：73, SEQ ID NO：One kind in 74
Or it is a variety of.
2. a kind of parasite detection kit according to claim 1, it is characterised in that：In step A-K, expand for detecting
The Leishmania of increasing or the nucleic acid probe composition of trypanosome DNA fragmentation include：SEQ ID NO：9, SEQ ID NO：14, SEQ ID
NO：77, SEQ ID NO：29, SEQ ID NO：30, SEQ ID NO：37, SEQ ID NO：52, SEQ ID NO：55, SEQ ID
NO：61, SEQ ID NO：62, SEQ ID NO：65, SEQ ID NO：69, SEQ ID NO：72.
3. a kind of parasite detection kit according to claim 2, it is characterised in that：Label examination is detected with nucleic acid probe
Agent box, nucleic acid probe be include nucleic acid sequence SEQ ID NO：76 oligonucleotides.
4. a kind of parasite detection kit according to claim 1, it is characterised in that：Further include amplifing reagent, reagent,
One or more recombinases, endonuclease, an excision enzyme, single-stranded DNA binding protein or strand displacement polymerase.
5. a kind of parasite detection kit according to claim 4, it is characterised in that：Exonuclease is Exonucleolytic
Enzyme III, endonuclease are a kind of endonuclease IV.
6. a kind of parasite detection kit according to claim 1, it is characterised in that：Including detectable at least one mark
The nucleic acid sequence of the oligonucleotides nucleic acid probe of note：SEQ ID NO：9, SEQ ID NO：14, SEQ ID NO：77, SEQ ID
NO：29, SEQ ID NO：30, SEQ ID NO：37, SEQ ID NO：52, SEQ ID NO：55, SEQ ID NO：61, SEQ ID
NO：62, SEQ ID NO：65, SEQ ID NO：69, SEQ ID NO：72, SEQ ID NO：76.
7. a kind of detection method of parasite detection kit, it is characterised in that：Including using the parasite of claim 1 to detect
One or more nucleic acid amplification primers are to carrying out nucleic acid amplification reaction, and the spy by detecting nucleic acid amplification primers pair in kit
Specific amplification detects the presence of the DNA of parasite.
8. a kind of detection method of parasite detection kit according to claim 7, it is characterised in that：Nucleic acid is detected to expand
The method for increasing the specific amplicon of primer pair includes the amplification of nucleic acid combined with the nucleic acid probe comprising following nucleic acid sequence：
SEQ ID NO：9, SEQ ID NO：14, SEQ ID NO：77, SEQ ID NO：29, SEQ ID NO：30, SEQ ID NO：37,
SEQ ID NO：52, SEQ ID NO：55, SEQ ID NO：61, SEQ ID NO：62, SEQ ID NO：65, SEQ ID NO：
69, SEQ ID NO：72, SEQ ID NO：76.
9. a kind of detection method of parasite detection kit according to claim 7, it is characterised in that：Further include being formed
Include the mixture of the biological sample of at least one primer pair and participation amplified reaction, biological sample is body fluid or tissue sample.
10. a kind of detection method of parasite detection kit according to claim 7, it is characterised in that：Amplified reaction
It is the isothermal amplification of a recombinase polymeric enzymatic amplification.
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| CN111876512A (en) * | 2020-07-29 | 2020-11-03 | 深圳市疾病预防控制中心 | Reagent and kit for detecting two leishmania by isothermal amplification and application of reagent and kit |
| CN113755620A (en) * | 2021-08-30 | 2021-12-07 | 江苏省血液中心 | Nucleic acid detection kit for rapidly detecting two leishmania and application thereof |
| CN113817806A (en) * | 2021-09-28 | 2021-12-21 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Reagent and kit for isothermal amplification detection of trypanosoma cruzi and application of reagent and kit |
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